Anomalous signals within proteins can be used to derive phase information for crystal structure determination by single wavelength anomalous dispersion (SAD) experiments (Hendrickson and Teeter (1981) “Structure of the hydrophobic protein crambin determined directly from the anomalous scattering of sulfur” Nature 290:107-113 and Debreczeni et al. (2003) “In-house measurement of the sulfur anomalous signal and its use for phasing” Acta Crystallogr D 59:688-696). Unfortunately, the weak anomalous signals derived from sulfur or other atoms present in proteins result in the stringent need for highly redundant data (Dauter et al. (1999) “Can anomalous signal of sulfur become a tool for solving protein crystal structures?” J Mol Biol 289:83-92), which has limited the use of in-house SAD phasing. Heavy atoms, such as U, Ba, Xe, Te, and I, have strong anomalous signals at the CuKα wavelength, but it is generally difficult to place them at precise positions in a protein.
The anomalous signal (δf″) of iodine at the CuKα wavelength typically used with in-house generators (1.5418 Å) is 6.85e−, six times of that of selenium (1.14e−) and twelve times of that of sulfur (0.56e−) (Dauter et al. (2002) “Jolly SAD” Acta Crystallogr D 58:494-506). Therefore, the selective introduction of an iodine atom into proteins can reduce the high data redundancy and high-solvent content necessary for selenium or sulfur phasing (Dauter et al. (1999) J Mol Biol 289:83-92). The genetic incorporation of heavy atoms as described herein (e.g., genetic incorporation of iodoPhe) offers a number of advantages over current approaches for introducing heavy atoms for SAD phasing. One such method involves substituting bound water molecules at the surface of the protein with either halides or metal ions by soaking the crystal in a solution containing the relevant ions (Dauter et al. (2000) “Novel approach to phasing proteins: derivatization by short cryo-soaking with halides” Acta Crystallogr D 56(Pt 2):232-237 and Nagem et al. (2001) “Protein crystal structure solution by fast incorporation of negatively and positively charged anomalous scatterers” Acta Crystallogr D 57:996-1002). Unfortunately, this approach can produce a number of low occupancy sites whose positions must be determined before phases can be derived. Another method, telluromethionine (TeMet) incorporation (Boles et al. (1994) “Bio-incorporation of telluromethionine into buried residues of dihydrofolate reductase” Nat Struct Biol 1:283-284 and Budisa et al. (1997) “Bioincorporation of telluromethionine into proteins: a promising new approach for X-ray structure analysis of proteins” J Mol Biol 270:616-623), provides a significant anomalous signal at the CuKα wavelength (δf″=6.4 e−), but is limited by the extreme sensitivity of TeMet to oxidation, toxicity to the host organism, and difficulty in achieving quantitative replacement of Met with TeMet.
Among other benefits, the present invention provides compositions and methods that overcome the above noted difficulties by enabling site-specific, high efficiency incorporation of heavy atoms into proteins. For example, by genetically encoding iodoPhe with a unique codon, tRNA, and aminoacyl-tRNA synthetase, this amino acid can be quantitatively and efficiently incorporated at any desired site in a protein. Furthermore, the substitution of large hydrophobic residues with iodoPhe, at either surface or internal sites, is likely to cause minimal perturbation of the protein structure. A complete understanding of the invention will be obtained upon review of the following.